The magnitude of nitric oxide (NO) production by mammalian cells is dependent on numerous factors. Cells stimulated make inducible (inflammatory) NOS (iNOS) generate 100-to 1000-fold more NO than do cells containing distinct NOS isoforms present constitutively, and this high-output NO plays pathophysiological roles in inflammation and host defense. The principal objective of the proposed research is to elucidate the multiple mechanisms that are involved in the regulation or modulation of the high-output production of NO in vascular smooth muscle, endothelial cells and macrophages. The rationale for this objective is that in view of the widespread pathophysiological actions of iNOS-generated NO, it is essential to understand the mechanisms by which both iNOS and NO production are regulated. The central hypothesis that drives this proposal is that the production of both iNOS protein and NO is sensitive to regulation at various levels including NF-kappaB- mediated transcription, post-transcriptional stability of iNOS mRNA, post-translational stability of iNOS protein, and arginine availability as a substrate for iNOS. Four specific aims are proposed to achieve the objective: (a) to elucidate the mechanism by which LPS causes downregulation and degradation of iNOS in vascular smooth muscle cells and macrophages, (b) to elucidate the mechanisms by which NO elicits positive and negative feedback modulation of NF-kappaB activation of iNOS induction in vascular cells and macrophages, (c) to determine how LPS-induced arginase plays a role in regulating high-output NO production by INOS in vascular cells and macrophages and (d) to ascertain the novel role of Ng- Hydroxyarginine as a distinct biological effector molecule in regulating intracellular arginine levels and high-output NO production in vascular cells and macrophages. The proposed research represents a continuing long-term effort to understand the regulation of NO production and action in vascular cells and macrophages.